Rapid Preparation of Unsheathed Bacterial Flagella
未出鞘细菌鞭毛的快速制备
发布: 2015年03月20日第5卷第6期 DOI: 10.21769/BioProtoc.1425 浏览次数: 10468
评审: Fanglian HeAmit DeyEsteban Paredes-Osses
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参见作者原研究论文
The authors used this protocol in:
Dec 2013
Abstract
The flagellum is required for bacterial swimming and swarming motility. In the biphasic Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellar filament is build up by two distinct monomeric subunits, flagellin FliC and FljB. S. Typhimurium has the ability to switch between two flagellins, FliC and FljB, in a phase-variable manner. The switch to FliC is called phase H1 and considered important for bacterial growth and survival in the spleen in a murine infection model of typhoid fever. Flagellin is secreted as monomeric subunits, but the majority of flagellin is polymerized upon secretion as the flagellar filament. Salmonella flagellin has traditionally been isolated through a process involving multiple steps of centrifugation and acid treatment. Here, we delineate a simplified protocol for preparing Salmonella´s flagellin for analytical purpose to determine the amount of flagellin without the aid of antibodies. The growth conditions used were stationary phase, logarithmic phase and a low oxygen and high salt condition mimicking the gastrointestinal tract. Flagellin expression of other source organisms, such as other serovars of Salmonella enterica and Escherichia coli, including flagellar phase- or genetic variants can be analysed. Flagellin expression analysis complements flagella-associated phenotype analysis such as swimming and swarming behaviour.
Keywords: Bacteria (细菌)
Salmonella (沙门氏菌)
Flagellin (鞭毛蛋白)
Flagellar phase variation (鞭毛的相位变化)
Materials and Reagents
- Bacterial strains (S. Typhimurium)
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- 100% trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
- β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
- Trizma® base (powder) (Sigma-Aldrich, catalog number: T1503 )
- 1 M HCl solution (Sigma-Aldrich, catalog number: H1758 )
- Glycerol 1 L (Sigma-Aldrich, catalog number: 49781 )
- Sodium dodecyl sulfate - 500 g (Sigma, catalog number: L4390 )
- Tryptone (BD-catalog number: 211705 )
- Yeast extract (BD, catalog number: 212750 )
- Agar (BD, catalog number: 281230 )
- Bromophenol blue (Shelton scientific, catalog number: IB74040 )
- Glycerol stock for one vial (see Recipes)
- LB medium (see Recipes)
- 1 M Tris (see Recipes)
- 2x SDS sample buffer (see Recipes)
Equipment
- Falcon tubes (50 ml and 15 ml) (TPP, catalog numbers: 91050 and 91115 )
- Filter Flasks (pore size 0.22 μm diameter) (TPP, catalog number: 99500 )
- Eppendorf tubes (1.5 ml) (Sarstedt, catalog number: 72.690.001 )
- Incubator (adjusted to 37 °C) (Memmert, model: ULE500 )
- Rotary shaker (HTINFORS, model: Minitron )
- Spectrophotometer (Hitachi, model: U1100 )
- Thermoblock (PEQLAB, model: 1202 )
- Vortex mixer (VWR International, model: 1719 )
- Luer lock tips (BD PLASTIKPAK, catalog number: 302187 )
- Needle (0.51 mm outer diameter) (BD Microlance sterile, model25 g x 5/8” x 100)
- Sterile petri dishes (90 mm) (Sartorius, catalog number: 14-555-735 )
- Centrifuge (HERAEUS, model: PICO17 )
- Microcentrifuge (NeoLab, model: microcentrifuge II )
- pH meter (Hanna instruments, model: pH211 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Guyon, S. L., Rhen, M. and Römling, U. (2015). Rapid Preparation of Unsheathed Bacterial Flagella . Bio-protocol 5(6): e1425. DOI: 10.21769/BioProtoc.1425.
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分类
微生物学 > 微生物细胞生物学 > 细胞器分离
微生物学 > 微生物-宿主相互作用 > 细菌
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